ABSTRACT In eukaryotes the ATP dependent protein degradation by the ubiquitin-proteasome pathway removes short lived signaling protein that is critical in regulation of cellular process, degrades misfolded and damaged proteins whose accumulation is toxic to the cell and breaks down foreign proteins to generate antigenic peptides for presenting to the immune system. It is fundamental in understanding the mechanism of many human diseases, especially cancer and neurodegenerative diseases, e.g. Huntington disease. The eukaryotic 26S proteasome is formed by a 20S proteasome with the proteolytic active sites sequestered inside it and two 19S regulatory particles each contain six ATPases in contact with the 20S. A key role of the ATPases is to open the gated channel in the 20S to facilitate substrates enter for destruction. An important question in proteasome biology is that how short peptides of proteolytic products are released efficiently from CP to ensure a continuous substrate entering and products release required for the degradation of large protein substrates. A widely accepted yet untested paradigm is that the 26S proteasome functions unidirectional in which unfolded substrates enter the CP from one end and the proteolytic products exit from the opposite end. Another important question is what is the role of ATP hydrolysis by Rpt subunits during the Rpt ring assembly, and if the assembly requires CP as a template? In this application, we aim to address these questions. We will use near atomic resolution single particle cryoEM as our main structural analysis tool, together with other methods in molecular biology, biochemistry and biophysical tool, to elucidate the mechanisms that regulates the asymmetrical functionality of the symmetrical protein degradation machinery. The specific aims are (1) determine the mechanism of proteolytic products releasing from the 20S degradation chamber, (2) determine mechanism that coordinates the functions of proteasomal activators bound to the opposite ends of 20S core particle, and (3) determine the role of ATP hydrolysis in the assembly pathway of eukaryotic proteasomal ATPases. Substantial completion of these aims will advance our knowledge about the proteasome-mediated protein degradation that plays a key role in the pathogenesis of many human diseases.